Inclusion and exclusion criteria
Included were 31 HTx recipients >1 year post-transplant. Excluded were HTx recipients with (1). histologic evidence of allograft rejection graded ≥3 according to Texas classification (16) because acute allograft rejection affects diastolic function (11); (2) LV echocardiographic EF <55%, and ≥ moderate aortic or mitral valvulopathy; (3) LV end-diastolic pressure (LVEDP) ≥20 mmHg; (4) history of uncontrolled systolic arterial hypertension with >150 mmHg on chart review; (5) heart rate <55 and >120 bpm or pace maker treatment and (6) more severe transplant vasculopathy or coronary artery lesions requiring intervention.
Patients were included after informed consent was obtained. The study was approved by the local institutional review committee (protocol number 708). Endomyocardial layers from septal or LV myocardial specimens were obtained from explanted non-failing human hearts with EF ≥55% that were not eligible for organ transplantation because of donor age (n = 3), viral infection or blood group mismatch.
Assessment of cardiac function
Transthoracic echocardiography: Examination was performed with each participant in left lateral recumbent position using 2–5 MHz transducers and standard equipment [Acuson® Aspen, Sequoia, XP128 (Acuson, Malvern, PA, USA)]. IVRT was measured as the time from aortic closure to opening of the mitral valve with the continuous-wave Doppler positioned in the four-chamber view between the anterior mitral valve leaflet and the aortic valve. Echocardiograms were recorded for off-line analysis, measurements were the mean of three consecutive heart cycles.
Cardiac catheterization: Endomyocardial biopsies were taken from the right ventricular aspect of the interventricular septum under fluoroscopic control. LVEDP, aortic end-systolic and end-diastolic pressures were measured with fluid-filled catheters connected to Statham transducers. Hemodynamic measurements were determined as mean of three consecutive sinus beats. End-diastolic volume and EF were calculated by the centerline method.
Endomyocardial biopsies, mRNA isolation and real-time PCR
Endomyocardial biopsies: Specimens for gene expression analysis were obtained together with the routine biopsies when the patients were hospitalized for the annual post-transplant routine control including left and right heart catheterization and transthoracic echocardiography. Endomyocardial specimens were frozen in liquid nitrogen immediately after removal. mRNA isolation from microbiopsies was similar to Hullin et al. (17).
Gene expression in interventricular septum and free LV wall: Layers of 2 mm thickness were prepared from the trabeculated endomyocardial regions of the interventricular septum and the free LV wall of non-failing human hearts. Biomagnetic separation was used for mRNA isolation (17).
Quantitative gene expression: Coding sequences were cloned by RT-PCR with specific 5′- and 3′-oligonucleotide primers and reverse transcribed mRNA isolated from human non-failing LV (14). Amplified fragments were subcloned into pCR2.1-TOPO (Invitrogen®, Carlsbad, CA, USA), subcloned fragments were sequenced on both strands (MWG Biotech®, Fbersberg, Germany) and used for real-time PCR when identical to their respective GenBank sequences. Quantification in real-time PCR used standard curves amplified with 103–107 plasmids containing the subcloned coding sequences. Always, 2 biopsies/patient were measured as duplicates. For real-time PCR the iQ™ Supermix was used in the presence of a Taqman-probe; in the absence of a Taqman-probe the iQ™ SYBR Green Supermix (Bio-Rad® Laboratories, Hercules, CA, USA) was used, always followed by melt curve analysis (70°C–94°C at 0.5°C steps). Reactions were carried out on the iCycler iQ® Instrument (Bio-Rad® Laboratories AG). Concentrations of primer/TaqMan® probes were 2 μM always. Gel electrophoresis of real-time PCR reactions always visualized one single amplification product band.
Cardiac calsequestrin: 5′-primer: 5′- CTGAGCATCCTGTGGATCGAC; 3′-primer: 5′- TGTGGCCTGAATAGGTCAATCTT; positions on GenBank D55655: 1010-1030, 1098-1076. Amplification protocol: 94°C, 3 min; 57.8°C and 94°C, 30 and 20 s, 40 times; hold 25°C. Correlations: 0.997–0.999, efficiencies: 90.1–97.5%.
Phospholamban: 5′-primer: 5′- TTCTCTCGACCACTTAAAACTTCA, 3′-primer: 5′- CTGAGCGAGTGAGGTATTGGA, TaqMan® probe: 5′-6FAM- CTTCCTGTCCTGCTGGTATCATGG-Dabcyl; positions in GenBank M63603: 136–159, 202–192, 162–185. Protocol: 94°C, 3 min; 57°C and 94°C, each 30 sec, 40 times; hold 25°C. Correlations: 0.997–0.999, efficiencies: 92.3–102.1%.
Sarcoplasmic Ca2±-ATPase (SERCA2a): 5′-primer: 5′- TGAACCCTCCCACAAGTCTAAA, 3′-primer: 5′- CCAATCTCGGCTTTCTTCAGAG, TaqMan® probe: 5′-6FAM- CATCGTTCACGCCATCGCCAGTCA-Dabcyl; positions in GenBank M23115: 2037-2058, 2150-2129, 2122-2099. Protocol: see PLB. Correlations: 0.991–0.995, efficiencies: 92.5–106.8%.
Ryanodine receptor type 2 (RyR): 5′-primer: 5′- AAAGCCGAGGGAGAAGATGGA, 3′-primer: 5′- TCTGTTGATATGCTATGATTTTCTTCCA; positions on GenBank X98330: 13442-113462, 13587-13562. Protocol: 94°C, 3 min; 57°C 30 s, 72°C 20 s, 94°C 30 s, 40 times; hold 25°C. Correlations: 0.998–1.0, efficiencies: 90.9–91.8%.
Cardiac sodium-calcium exchanger (NCx): 5′-primer: 5′- AGACCTTCTTCCTTGAGATTGGA, 3′-primer: 5′- GCTCATTCAATAACAGGGCTTTC, TaqMan® probe: 5′-6FAM- TCTCACTCATCTCCACCAGGCG-Dabcyl; positions in GenBank M91368: 1939–1961, 2013–1991, 1989–1968. Protocol: see PLB. Correlations: 0.990–0.996, efficiencies: 94.2–104.6%.
Desmin: 5′-primer: 5′- TGACCGCTTCGCCAACTA; 3′-primer: 5′- GAGTAGCTGCATCCACGTCC; positions on GenBank U59167: 428–445, 735–716. Protocol: 94°C, 3 min; 56°C and 94°C, 30 and 20 s, 40 times; melt curve, hold 25°C. Correlations: 0.994–0.996, efficiencies: 94–99.1%.
Collagen I: 5′-primer: 5′- AAGAGGAAGGCCAAGTCGAG; 3′-primer: 5′- GATCACGTCATCGCACAACA; TaqMan® probe: 5′-6FAM- TCACCTGCGTACAGAACGGCCT-Dabcyl; positions on GenBank NM_000088: 187-206, 341-–322, 232-253. Protocol: 94°C, 3 min; 56°C, 72°C, 94°C, 20, 30, 20 s respectively, 40 times; hold 25°C. Correlations: 0.991–0.999, efficiencies: 90–98.1%.
Normalization: Because of the unchanged gene expression of cardiac calsequestrin in the non-failing and failing human heart (17,14), its gene expression was assessed in each endomyocardial biopsy. Normalization was performed dividing the cardiac calsequestrin gene expression in the particular biopsy by the mean of cardiac calsequestrin gene expression in all biopsies.
Collagen content in biopsies was estimated by color segmentation of digital images. The imaging software (Image-Pro Plus 5.1, MediaCybernetics™, Silver Spring, MD, USA) recognizes particular colors and distinct shades and digitally highlights pixels of a particular color or shade specified within the field. Fibrosis becomes blue with trichrome stain and is highlighted based on the operator's threshold settings allowing for calculation of the amount of area occupied by fibrosis within the field.
Verification of normal distribution of data was accomplished with visual inspection of histograms. Power transformations were used to normalize skewed distributions. Student's t-test for comparison of gene expression in endomyocardial layers. t-test, chi square test, Spearman rank correlation were used where appropriate. All tests were computed using the Stat View 4.57 software (Abacus, Inc., Berkeley, CA, USA).